The adrenal cortex of the guinea pig contains specific steroid- binding proteins which have been only partially purified and characterized; their function is as yet undetermined. For instance, there are proteins which specifically bind cholesterol, cholesteryl sulfate, pregnenolone , and pregnenolone sulfate. These proteins are of great interest because the rate-limiting reaction in steroido-genesis is the conversion of cholesterol to pregnenolone or cholesteryl sulfate to pregnenolone sulfate. The pregnenolone-binding protein has been determined to behave as a 58 kD protein by gel permeation chromatography and as a 34 kD species by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 34 kD protein was electroeluted from acrylamide gels and injected into rabbits; an antibody was produced that specifically bound to and removed from solution pregnenolone-binding activity. When antibody-bound protein was eluted from the protein A-IgG column and re-examined by SDS- PAGE, a 34 kD protein was again generated. As a control, a 29 kD protein was simultaneous electroeluted and antibodies were raised against it. Unlike the situation with antibody to the 34 kD protein, antibody to the 29 kD protein did not interact with pregnenolone-binding activity. These results confirm that the pregnenolone-binding protein is a 34 kD protein as previously reported. Unfortunately, antibody titers to date are low and this has impeded progress, but efforts are being made to remedy this situation. In addition to the non-catalytic steroid-binding proteins, efforts are underway to isolate and produce antibodies to the cytochrome P450 for cholesterol side-chain cleavage. The latter antibody will be used to quantitate P450 in the outer and inner adrenocortical zones of the guinea pig. In this animal model cholesterol side-chain cleavage activity is stimulated in the outer zone but not the inner zone. The antibody to P450scc will also be used to isolate the mRNA for P450scc.